Electrochromatography A Hybrid Separation Technique Gel Filtration Chromatography

Electrochromatography - A Hybrid Separation Technique Gel Filtration Chromatography + Capillary Electrophoresis = Electrochromatography [info shamelessly taken from Wikipedia and http: //www. unimicrotech. com/products_CEC_instrument. htm] Wilkes University -CHM 342

The Idea n n n Combine the attributes of size exclusion chromatography (gel filtration chromatography) with the benefits of gel electrophoresis. The two separation mechanisms both operate along the length of a gel filtration chromatography column which has an electric field gradient applied to the column. Useful for the separation of large biomolecules q q q separated by size due to the gel filtration mechanism separated by electrophoretic mobility (gel electrophoresis) Also other chromatographic solute retention mechanisms Wilkes University - CHM 341

The Basics - Gel Filtration or Permeation n Size exclusion chromatography (SEC) q q q n particles are separated based on hydrodynamic volume aqueous mobile phase = gel filtration chromatography organic mobile phase = gel permeation chromatography widely applied for purification and analysis of synthetic or bio-polymers (proteins, polysaccharides, & nucleic acids) q q q biopolymers - use a gel stationary phase (usually polyacrylamide, dextran, or agarose) at low pressures synthetic polymers - use either a silica or crosslinked polystyrene stationary phase at higher pressures Various mobile phases can be used Wilkes University - CHM 341

The Basics – Hydrodynamic Volume n n n Related to the radius of gyration - measure of the size of an object q calculated as the r. m. s. distance of the parts (or surface) of an object from either its center of gravity or an axis the radius of gyration is used to describe the dimensions of polymer chains chain conformations of polymer samples are quasi infinite, change over time q the "radius of gyration" discussed in polymer physics must usually be understood as a mean over all polymer molecules of the sample and over time q Rg determined experimentally with static light scattering as well as with small angle neutron- and x-ray scattering. q The hydrodynamic radius is numerically similar, and can be measured with size exclusion chromatography. Wilkes University - CHM 341

SEC – Illustrated Wilkes University - CHM 341

Gel Filtration or Permeation – Inst. n HPLC type setup q q q q Controller Injector Liquid mobile phase High pressure pumps column (“size exclusion” stationary phase) Detector (UV, fluor. , or other) “collector” (as waste or fractions) Data system (PC) Wilkes University - CHM 341

Standard Gel Electrophoresis n Separation uses a “gel" as the stationary phase – it is often a crosslinked polymer q q n For proteins or small nucleic acids (DNA, RNA, or oligonucleotides) the gel is usually composed of acrylamide and a cross-linker (in various ratios) producing mesh networks of polyacrylamide with different sized pores. For larger nucleic acids (greater than a few hundred bases), agarose is the preferred matrix. "Electrophoresis" refers to the electromotive force (EMF) that is used to move the molecules through the gel matrix. q q q the molecules move through the matrix at different rates, usually determined by mass, Motion is toward the positive anode if negatively charged or toward the negative cathode if positively charged Wilkes University - CHM 341

The Basics – Cap. Electrophoresis n Capillary electrophoresis (CE), also known as capillary zone electrophoresis (CZE) q q q used to separate ionic species by their charge and frictional forces. traditional electrophoresis, electrically charged analytes move in a conductive liquid medium under the influence of an electric field Introduced in the 1960 s, the technique of capillary electrophoresis (CE) was designed to separate species based on their size to charge ratio in the interior of a small capillary filled with an electrolyte Wilkes University - CHM 341

The Basics – Electrophoretic Mobility n n analyte electrophoretic migration velocity (up) toward the electrode of opposite charge is: u p = μ p. E q q n μp = electrophoretic mobility E is the electric field strength electrophoretic mobility at a given p. H q q q z is the net charge of the analyte the viscosity (η) of the medium r is the Stokes radius of the analyte n D is the diffusion coefficient. Wilkes University - CHM 341

The Basics – electroosmotic flow n n EOF does not significantly contribute to band broadening as in pressure-driven chromatography. Capillary electrophoresis separations can have several hundred thousand theoretical plates Wilkes University - CHM 341

The Basics – electroosmotic flow n n electroosmotic flow (EOF) of buffer is directed toward the cathode (-) the electroosmotic flow of buffer > electrophoretic flow of the analytes all analytes are carried along with the buffer toward the cathode analytes do migrate toward the electrode of opposite charge q q n negatively charged analytes attracted to anode (+), counter to the EOF positively charged analytes attracted to cathode (-) with the EOF anionic analytes retained longer due to conflicting electrophoretic mobilities q small multiply charged cations migrate quickly and small multiply charged anions are retained strongly Wilkes University - CHM 341

The Instrumental Requirements n Capillary Electrophoresis Wilkes University - CHM 341

Electrochromatography n n high efficiency of CE is combined with the high selectivity of micro-HPLC hybrid technique known as capillary electrochromatography (CEC). q q utilizes columns similar to those used in micro-HPLC the mobile phase is driven by an electric potential as in CE separation mechanism is the result of the combination of chromatographic partitioning and electrophoretic migration. CEC can be done in a CE instrument with a micro. HPLC column Wilkes University - CHM 341

Electrochromatography Wilkes University - CHM 341

Electrochromatography n Fast separation of 16 EPA priority pollutants. Column: EP-100 -201. 5 -C 18 (1. 5 mm non-porous ODS, Micra Scientific, Inc. , Northbrook, IL). Mobile phase: 70% CH 3 CN in 30% 2 m. M TRIS. Voltage: 55 k. V. Injection: 5 k. V/2 s. Detection: LIF, ex: 257 nm, em: 400 nm. Wilkes University - CHM 341

Gradient Electrochromatography Wilkes University - CHM 341

Gradient Electrochromatography n n n Separation of 16 PAHs Column: EP-75 -26 -3 -C 18. Voltage: 20 k. V for the isocratic separations. Injection: 5 k. V/5 s. Detection: LIF, ex: 257 nm, em: 400 nm. Sample: 1. naphthalene, 2. acenaphthylene, 3. acenaphthene, 4. fluorene, 5. phenanthrene, 6. anthracene, 7. benzo[b]fluoranthene, 8. pyrene, 9. benz[a]anthracene, 10. chrysene, 11. benzo[b]fluoranthene, 12. benzo[k]fluoranthene, 13. benzo[a]pyrene, 14. dibenz[a, h]anthracene, 15. benzo[ghi]perylene, and 16. indeno[1, 2, 3 -cd]pyrene. Wilkes University - CHM 341